Prospective identification of cross-reactive human peptide-MHC ligands for T cell receptor based therapies

T cell receptor (TCR)-based therapeutic cells and agents (e.g. adoptive T cell, TCR-engineered T cells, ImmTACs, TCR mimic antibodies, neoantigen vaccines) have emerged as a new class of effective and selective cancer therapeutics against intracellular cancer-associated proteins. These agents rely on presentation of short peptides derived from cellular, viral or phagocytosed proteins on major histocompatibility complex (MHC). However, cross-reactivities of these agents to off-target cells and tissues are poorly understood, difficult to predict, and have resulted in serious, sometimes fatal, adverse events. Here we have developed a mammalian, minigene-based method (termed PresentER) that encodes MHC-I peptide ligands for functional immunological assays as well as for determining the reactivities and potential cross-reactivities of TCR-like therapeutic drugs against libraries of MHC-I ligands. This system is highly specific to, and entirely dependent on, the genetically encoded MHC peptide sequence, because it does not require proteasome cleavage, transporter associated with antigen processing (TAP) or processing, for immune presentation. For the expression of defined MHC-I ligands, this system is superior to expression of full-length cDNA. PresentER-expressing cells can be bound by TCR and TCR mimic (TCRm) antibodies, activate antigen-specific T cells, lead to antigen-specific cell death in vitro and tumor rejection in wild-type mice. Using PresentER in a pooled library screen, we find dozens of MHC-I ligands encoded in the human proteome that are cross-reactive with two TCR mimic antibodies and are not predictable by other methods. PresentER has broad and immediate utility for both basic and translational studies in immunology and oncology.